CVAK104 Is a Novel Poly-L-lysine-stimulated Kinase
That Targets the ?2-Subunit of AP2*
Received for publication, March 4, 2005
Published, JBC Papers in Press, April 4, 2005, DOI 10.1074/jbc.M502462200
Sean D. Conner‡§ and Sandra L. Schmid¶?
From the ‡Department of Genetics, Cell Biology, and Development, The University of Minnesota, Minneapolis, Minnesota
55455 and ¶Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037
Isolated clathrin adaptor protein (AP) preparations
are known to co-fractionate with endogenous kinase ac-
tivities, including poly-L-lysine-stimulated kinases that
target various constituents of the clathrin coat. We have
identified CVAK104 (a coated vesicle-associated kinase
of 104 kDa) using a mass spectroscopic analysis of adap-
tor protein preparations. CVAK104 is a novel serine/
threonine kinase that belongs to the SCY1-like family of
protein kinases, previously thought to be catalytically
inactive. We found that CVAK104 co-fractionates with
adaptor protein preparations extracted from clathrin-
coated vesicles and directly binds to both clathrin and
the plasma membrane adaptor, AP2. CVAK104 binds
ATP, and kinase assays indicate that it functions in vitro
as a poly-L-lysine-stimulated kinase that is capable of
autophosphorylation and phosphorylating the ?2-adap-
tin subunit of AP2.
Endocytosis involves the invagination of specialized regions
of the plasma membrane, which pinch off to form cargo-con-
taining vesicles that are transported into the cell. Although
several diverse endocytic pathways are present in eukaryotic
cells, the clathrin-mediated pathway is ubiquitous and the
most efficient mechanism. This internalization pathway is key
to a variety of biological processes that range from the down-
regulation of activated signaling receptors to synaptic vesicle
Clathrin-mediated endocytosis is a dynamic process that is
tightly regulated both spatially and temporally and involves
the coordinated effort of a host of cytosolic and membrane-
associated proteins (2, 3). Central to endocytic vesicle forma-
tion are two protein complexes, clathrin and AP2,1which make
up the major coat constituents. The formation of progressively
curved clathrin lattices on the cytoplasmic face of the plasma
membrane is believed to be a driving force in the generation of
coated pits and coated vesicles. However, clathrin, being inca-
pable of directly binding to the plasma membrane or to cargo,
requires the action of AP2. AP2 is a multifunctional complex
consisting of two large peptide chains (? and ?2), a medium
(?2), and a small chain (?2) that coordinates the recruitment
and assembly of clathrin and links it to cargo that is marked for
internalization. AP2 also acts as a platform to recruit other
functionally relevant endocytic factors, such as amphiphysin,
eps15, epsin, AP180, AAK1, etc. (4–6).
Although the mechanisms involved in regulating the clath-
rin-mediated endocytic pathway are not fully understood, ac-
cumulating evidence suggests that phosphorylation cycles may
be a key step (7–9). Indeed, phosphorylation of the clathrin
heavy chain (10) and the large and medium subunits of AP2
(11–13) have been demonstrated to be important to the clath-
rin-mediated internalization of growth factors and nutrients,
respectively. However, the phosphorylation of endocytic factors
is not limited to the major coat constituents. Many other com-
ponents of the endocytic machinery (e.g. dynamin 1, amphiphy-
sin 1 and 2, synaptojanin, AP180, epsin, and eps15), collec-
tively known as the dephosphins, are phosphorylated in resting
neurons and then coordinately dephosphorylated following
nerve terminal stimulation (14).
Although it is clear that phosphorylation plays an important
role in clathrin-dependent internalization, a more detailed un-
derstanding of the endocytic regulatory mechanisms will re-
quire the identification of the respective kinases that target
endocytic components. Multiple kinases have been defined that
target the major constituents of clathrin-coated vesicles. Al-
though Src kinase (10) and casein kinase II (15) phosphorylate
the clathrin heavy and light chains, respectively, and two dis-
tantly related kinases, AAK1 (5) and cyclin G-associated
kinase/auxilin 2 (16, 17) phosphorylate the ?2-subunit of AP2,
the ?- and ?2-adaptin kinases remain to be determined.
We have taken a directed proteomic approach to identify
novel kinases associated with adaptor protein preparations.
Here we report the identification and initial characterization of
a novel serine/threonine kinase that we have named CVAK104
(coated vesicle-associated kinase of 104 kDa).
Reagents—Monoclonal antibodies against the clathrin heavy chain
(TD.1) and adaptins (100/1, 100/2, and 100/3) were generous gifts from
Dr. F. Brodsky (University of California at San Francisco, San
Francisco, CA) and Dr. E. Ungewickell (Hanover Medical School,
Hanover, Germany), respectively. Dr. Matthias Jost, in the Schmid
laboratory, generated polyclonal antibodies against a brain-specific
splice variant of ?-adaptin (amino acids 706–727, rabbit, number
3492) fused to GST as described previously (18). Bovine brain was
fractionated as previously described (5). Adaptor proteins and clath-
rin triskelia were isolated essentially as described previously (19, 20).
Kinase inhibitors included H89, staurosporine, roscovitine, and olo-
moucine (Calbiochem), protein kinase C/calmodulin kinase inhibitor
mixture, and cAMP-dependent protein kinase inhibitor peptide
(Upstate Biotechnology, Lake Placid, NY), heparin (average molecu-
lar mass, 15 kDa) and genistein (Sigma). Poly(Glu:Tyr) (4:1) peptides
(molecular mass, 20–50 kDa) were also from Sigma.
* The Scripps Research Institute manuscript number is 16923-CB.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
§ Supported by the Leukemia and Lymphoma Society (3160-5). To
whom correspondence should be addressed: The University of Minne-
sota, Dept. of Genetics, Cell Biology, and Development, 6–160 Jackson
Hall, 321 Church St. SE, Minneapolis, MN 55455. Tel.: 612-625-3707;
Fax: 612-625-4648; E-mail: email@example.com.
? Supported by National Institutes of Health Grant R37-MH61345.
1The abbreviations used are: AP, adaptor protein; GST, glutathione
S-transferase; PBS, phosphate-buffered saline; PBST, PBS containing
0.1% Tween 20; FSBA, fluorosulphonylbenzoyladenosine; AAK1, adap-
tor associated kinase I.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 280, No. 22, Issue of June 3, pp. 21539–21544, 2005
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
by guest on December 29, 2015
Sean D. Conner and Sandra L. Schmid
2005, 280:21539-21544.J. Biol. Chem.
2-Subunit of AP2
Poly-l-lysine-stimulated Kinase That
CVAK104 Is a Novel
Membrane Transport, Structure, Function,
doi: 10.1074/jbc.M502462200 originally published online April 4, 2005
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